Bacteria possess a remarkable system for translational quality control. In a process known as trans-translation, tmRNA enters stalled ribosomes and acts as a template, encoding a short peptide tag that marks the nascent polypeptide for destruction. The tmRNA-directed synthesis of a single polypeptide from two RNA templates presents challenges to our understanding of ribosome function. The objective of this proposal is to determine the mechanism by which tmRNA, its protein partner SmpB, and the ribosome clear away the stalled mRNA, license tmRNA entry, and position it properly to resume translation in frame. The power of genetic selections to rapidly assay large libraries of mutants (hundreds of millions) in a relevant in vivo context will be brought to bear on structure-function studies of these three components, clarification of interactions between them, and the identification of genes responsible for uncharacterized activities in trans- translation. The specific aims of the project are: 1) Using a novel genetic selection that ties the life of the cell to tmRNA function, identify tertiary structures and proximal sequences in tmRNA that ensure translation resumes at the correct triplet on the tmRNA template. 2) Adapt this positive selection to the study of SrnpB to determine sequences and structures responsible for tricking the decoding machinery into allowing tmRNA into the ribosome. The molecular interactions required for this activity will be identified by evolving genetic interactions that restore wounded SmpB function. 3) Using a genetic selection against tmRNA function (in which tagging causes cell death), identify the endonuclease that cleaves mRNA inside stalled ribosomes, clearing the way for tmRNA entry. It is our hypothesis that this endonuclease activity is a latent function of the ribosome itself. Elucidation of the mechanism of trans-translation will yield insight into important aspects of ribosome function in protein synthesis. The trans-translation machinery, found only in bacteria, may also serve as a target for future antimicrobials.